S. aureus is an important cause of biofilm-related infections such as endocarditis, chronic osteomyelitis, corneal infections involving lens implants, and medical device-related infections. Infections involving staphylococcal biofllms are often very difficult to treat with antibiotics and may be recurrent or require surgical removal of the infected device or tissue. Antibiotic susceptibility tests used in the clinical laboratory measure the resistance of planktonic or free-floating bacteria and do not account for the fact that S. aureus biofilms are significantly more resistant to most antibiotics than planktonic bacteria. The characteristics of biofilms that make them refractory to antibiotic therapy are not well understood at this time. The long-term objective of this project is to characterize the properties of staphylococcal biofilms that make them refractory to antimicrobial chemotherapy. The immediate goal of the proposed project is to assess the role in antibiotic resistance of poly-N-acetyl glucosamine (PNAG), which plays a critical role in S. aureus biofilm formation. First, the influence of PNAG expression on antibiotic resistance of planktonic bacteria will be characterized. Next, a PNAG-independent biofilm model, based on the cross linking of bacteria via protein A / Fc-receptor interactions, will be implemented so that the role of PNAG in biofilm antibiotic resistance can be separated from its role in biofilm formation. In addition, the effect of PNAG on the penetration of fluorescently labeled antibiotics throughout biofilms will be analyzed by confocal microscopy. Finally, the interaction between PNAG and antibiotics commonly used to treat S. aureus infections will be characterized. We hypothesize that PNAG plays an important role in the resistance of S. aureus biofilms by acting as a physical barrier against antibiotic penetration and expect that knowledge of the role of PNAG in S. aureus biofilm resistance to antibiotics will advance the development of therapies used to combat these infections.